*2.1. Patients*

We carried out a prospective study at the Gastroenterology Unit of "A.O.U. Città della Salute e della Scienza di Torino" hospital, Italy from April 2019 to October 2019.

Patients a ffected by IBS-D were recruited and treated with a strain-specific probiotic therapy for 8 or 12 weeks. Probiotic therapy consisted of daily administration of 1 × 10<sup>9</sup> colony-forming unit (CFU) of *B. longum* ES1 away from meals. Inclusion criteria were: age between 16 and 65 years old, diagnosis of IBS-D according to the Rome IV criteria and Bristol stool scale [28], body mass index (BMI) < 30 kg/m2, willingness to sign the informed consent to participate to the study. Exclusion criteria were: history of GI surgery, diagnosis of IBD or CD, thyroid diseases, diverticular disease, small intestine bacterial overgrowth (SIBO), colorectal cancer, other clinically relevant diseases, treatment with drugs that alter intestinal function (e.g., opiates, anticholinergics and laxatives), treatment with antibiotics (any previous antibiotic therapy should have been discontinued at least 4 weeks before the start of probiotic therapy) and other pre/probiotics (other pre/probiotics therapy should have been discontinued at least 2 weeks before starting therapy), pregnancy/breastfeeding.

Clinical history, data on physical examination, recent biochemical examinations and signed informed consent were collected. Disease severity was assessed at baseline with the Functional Bowel Disorder Severity Index (FBDSI) questionnaire [29]. At baseline (T0) and after 8 or 12 weeks of probiotic therapy (T1), patients were evaluated with Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) questionnaire for the clinical evaluation of disease [30] and Irritable Bowel Syndrome Quality of Life (IBS-QoL) questionnaire for life quality assessment [31]. IBS-QoL consists of 34 items and 8 domains; for the purpose of analysis, we considered the overall questionnaire score. The final raw scores of IBS-QoL were presented on a scale between 0 (poor quality of life) and 100 (maximum quality of life).

All patients underwent baseline venous sampling (T0) and after 8 or 12 weeks of therapy (T1); serum was collected in polypropylene 2 mL tubes labelled with the study participant identification code and stored at −80 ◦C until analysis.

The study followed the principles of the Declaration of Helsinki and was approved by the local ethics committee (Comitato Etico Interaziendale A.O.U. Città della Salute e della Scienza di Torino—A.O. Ordine Mauriziano—A.S.L. Città di Torino) (approval code 0056924).

#### *2.2. Measurement of Serum Zonulin and Cytokines*

Serum zonulin was assessed by competitive enzyme-linked immunosorbent assay (ELISA) (IDK ® Zonulin ELISA Kit, Immunodiagnostik AG, Bensheim, Germany) according to the manufacturer's instructions. Concentrations were calculated using a four-parameter algorithm, and the results were given in ng/mL, as previously reported [8]. The cytokine panel, including IL-6, IL-8, IL-10, IL-12p70, IL-23, IL-33, IFNγ and TNF α, was measured in serum samples by Bio-Plex ® Multiplex Immunoassay (Bio-rad Laboratories, Hercules, CA, USA) on a Luminex ® 200 system (Luminex Corporation, Austin, TX, USA). Individual standard curves were generated for each cytokine; the results are given in pg/mL [32]. Personnel performing laboratory investigations were blind to all the characteristics of the patients included in the study.
